Vector and host cellular biology in AAV vector transduction in vivo

腺相关病毒(AAV)是一种非致病性、复制缺陷病毒。重组AAV(RAAV) 显示了作为基因治疗的病毒载体的前景。RAAV具有一个由3个衣壳蛋白组成的简单结构 和单链DNA基因组，不表达病毒编码的蛋白质。尽管它不是致病的 和复制缺陷的性质，存在由病毒介导的细胞内病毒与宿主的相互作用 基因组DNA(即带有发夹的单链DNA)。尽管在这方面取得了实质性的进展 应用此载体系统治疗多种疾病，细胞内病毒-宿主相互作用的作用 RAAV载体生物学和体内宿主细胞生物学(在动物和人类中)在很大程度上仍不清楚。我们的 这项建议的最终目标是充分了解体内和细胞内复杂的rAAV载体生物学 转导组织中的体细胞生物学。为了实现这一目标，我们将具体集中在 RAAV基因组、宿主染色体DNA和宿主细胞DNA修复机制之间的相互作用。我们 最近发现，如果rAAV载体基因组反向末端重复序列(ITR)发夹结构不是 由特定宿主细胞的核酸内切酶活性决定的，一种不寻常的形式，无端双链 线性单体rAAV载体基因组，在小鼠的转导细胞中积累。基于这一观察， 我们假设，通过病毒-宿主细胞相互作用激活这种细胞内切酶活性 在rAAV载体基因组加工和体内稳定转导建立中的中心作用。在 建议，我们将利用这种不寻常的无末端载体基因组结构的形成来鉴定 参与载体-宿主细胞相互作用的细胞因素导致AAV-ITR发夹环打开(目标1)。 一旦我们确定了细胞因子，我们将研究细胞因子和载体基因组是如何在 以及rAAV载体如何激活该网络(目标2)。此外，我们还将调查rAAV是如何 载体基因组、宿主染色体DNA和细胞因素相互作用，产生载体基因组 体内各种组织的整合(目标3)。最后，因为这些细胞因子构成了DNA修复 因为DNA修复效率随着年龄的增长而降低，基因组的不稳定性随着年龄的增长而增加，我们将 研究年龄如何影响体内的媒介和宿主细胞生物学(目标4)。拟议的项目应 为建立成功的人类基因治疗的智力基础作出了重大贡献。

Adeno-associated virus (AAV) is a non-pathogenic, replication defective virus. Recombinant AAV (rAAV) shows promise as a viral vector for gene therapy. rAAV has a simple structure consisting of 3 capsid proteins and a single-stranded DNA genome, and does not express virally encoded proteins. Despite its nonpathogenic and replication defective nature, there are intracellular virus-host interactions mediated by viral genome DNA (i.e., single-stranded DNA with hairpins). Although there has been substantial progress in the application of this vector system to treat various diseases, the role of intracellular virus-host interactions in rAAV vector biology and host cellular biology in vivo (in animals and humans) remains largely unknown. Our ultimate goal in this proposal is to substantially understand complex rAAV vector biology in vivo and cellular biology of somatic cells in transduced tissues. Toward achieving this goal, we will specifically focus on interactions between rAAV genomes, host chromosomal DNA and host cellular DNA repair machinery. We recently discovered that, if rAAV vector genome inverted terminal repeat (ITR) hairpin structures are not resolved by a specific host cellular endonuclease activity(ies), an unusual form, no-end double-stranded linear monomer rAAV vector genomes, accumulate in transduced cells in mice. Based on this observation, we hypothesize that activation of this cellular endonuclease activity via virus-host cell interactions plays a central role in rAAV vector genome processing and establishment of stable transduction in vivo. In the proposal, we will take advantage of the formation of this unusual no-end vector genome structure to identify cellular factors involved in vector-host cell interactions leading to AAV-ITR hairpin loop opening (aim 1). Once we identify cellular factors, we will investigate how the cellular factors and vector genomes interact in a network and how this network is activated by rAAV vectors (aim 2). In addition, we will investigate how rAAV vector genomes, host chromosomal DNA and the cellular factors interact, resulting in vector genome integration in various tissues in vivo (aim 3). Finally, because such cellular factors constitute DNA repair machinery and because DNA repair efficacy decreases and genomic instability increases with age, we will investigate how age affects vector and host cellular biology in vivo (aim 4). The proposed project should substantially contribute to building an intellectual foundation for successful human gene therapy.